Team:Valencia UPV/Project/overview

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Pests cause great economical loses in agriculture and impedes an optimal use of the resources. Treatments used nowadays have some not-so-wanted faces: Pesticides do not target specifically enough to pests and cause alterations in other organisms, and chemical production of sex pheromones is expensive and generates contaminant by-products.

Sexy Plant rises as a pest control strategy based on the use of mating disruption to prevent laying. Mating disruption is based on the release of sex pheromones to the air, so male’s sex pheromones receptors are not able to trace females anymore. This strategy is effectively used in field already with chemically synthesised pheromones [1]. Our final goal is to increase the sustainability of this approach by intercropping few Sexy Plants emitting mating-disrupting pheromones.

Sexy Plant produces and releases in Nicotiana benthamiana several sex pheromones (Z11-16:OH, Z11-16:OAc and Z11-16:Ald) for a broad number of Lepidoptera species (moths). We implemented previous work on production pathways done by Ding et al. [2] into our system to produce the first two pheromones (Z11-16:OH and Z11-16:OAc). We knew about the importance of Z11-16:Aldehide to control a large number of species, so inspired by Hagström approach [3], we introduced Fatty Acid reductase to transform Z11-16:OH to Z11-16:Ald. As result, the sexy plant is able to produce three sex pheromones involved in moth’s mating disruption with 2 or 3 additional enzymes (Figure 1).

Biosafety is a major concern in our project. For that reason, we have developed a biosafety module which allows an easy identification of the plant by identity preservation and prevents the plant to spread its genetic material via pollen. Identity preservation is achieved by the production of a chromoprotein so that the plant acquires a differential colour. Dispersion of the genetic material is prevented by male sterility, as results of the tissue-specific expression of a RNAse (Barnase) in tapetum cells under the regulation of the promoter TA29 [4]. This way, autogamous Sexy Plants cannot be self-pollinate and seeds formation is blocked.

Following the principle of modularity in synthetic biology, Hagström et al [2] produced the insect pheromone Z-11:160H in yeast from palmitic acid by introducing a delta-11 fatty-acyl desaturase and a fatty-acyl reductase from Agrotis segetum. Recently, Ding et al [3] had already done the same work taking into account that palmitate is synthesised in the chloroplast, leading to successful results.

In Ding's work they use a delta 11 desaturase mRNA from Amyelois transitella (accession number JX964774); HarFAR_KKYR, an improved version of HarFAR-3 fatty acid reductase from Helicoverpa armigera (accession number JF709978) with an almost five folds conversion rate, and 1,2-diacyl-sn-glycerol:acetyl-CoA acetyltransferase from Euonymus alatus (accession number GU594061). With these three enzymes we would obtain two pheromones: Z-11:16OH and Z-11:16OAc.

Moreover, inspired by Hagström's discussion we also thought about using an alcohol oxidase in order to try to obtain pheromone Z11-16:Ald from Z11-16:OH. According to the SolGenomics database, there is already a fatty-acid alcohol oxidase in Nicotiana benthamiana but there was no certainty that the enzyme would work in in vivo conditions. On the other hand, we learned that Candida tropicalis had a fatty alcohol oxidase that catalysed the conversion from Z11-16:Ald to Z11-16:O [4,5,6,7]. (At the moment it is not clear from which organism will the fatty acid alcohol oxidase be obtained).

At the same time we were planning which pheromones could we produce, we also took into account on which insects will they have effect. We did a thorough search in the Pherobase database (http://www.pherobase.com) and we checked which insect met the conditions of having one of those as a major pheromone component and being a plague.

TRICHOMES

Once the metabolic pathway needed to synthetize the desired pheromone has been properly described and characterized, a method that allows pheromone diffusion from the plant to the exterior environment must be implemented. With this objective in mind, we decided to express the enzymes required to produce the pheromones specifically in glandular trichomes, which are secretory structures naturally present in the plant. For doing so, the enzyme coding sequence would be under the control of CSP2 promoter from Nicotiana tabacum [8], inducing their specific expression in trichomes and allowing the pheromones secretion.

SWITCH

The pheromone production would suppose an additional energy cost to the plant, therefore the objective is to activate this pathway and the subsequent pheromone production only when necessary, upon Lepidoptera mating season. The regulation mechanism chosen with that purpose has been the one naturally controlling the CUP1 metallothionein gene in S. cervisiae [9]. CUP1 function is to chelate copper ions to avoid its toxicity to the yeast; therefore it is only expressed under high copper concentration. This regulation mechanism relies on the expression of a transcription factor (ACE1 or CUP2) that changes its conformation under high copper concentrations allowing its interaction with a regulating region (Metal Responsive Element, MRE) in the promoter of the CUP1 gene.

Taking as a reference this mechanism, Mett et al adapted it so it could be used in whole plants. Takanori Saijo and Akitsu Nagasawa [10] posteriorly improved its efficiency. We decided to take their work as an example and use a system in which the transcription factor CUP2 or ACE1 is expressed under the constitutive promoter P35S, and the enzymes responsible for the pheromone production are expressed under the regulation of a inducible promoter, composed of the MRE domain fused with the P35S minimal promoter. Therefore, CUP2 transcription factor would only interact with MRE and the pheromone will be produced under high copper concentrations. A copper-rich solution will be applied to the plant by foliar spraying or soil drenching during Lepidoptera mating season allowing pheromone production on demand.

BIOSAFETY

One of the concerns about having transgenic plants in the field involves its uncontrolled spread. It is being developed a biosecurity module containing two parts: a male sterility submodule and an identity preservation submodule.

The male sterility submodule is based on a barnase, an RNase gene from Bacillus amyloliquefaciens, specifically expressed in anthers under the regulation of the TA29 tapetum-specific promoter. As result, pollen from these plants will not be capable of fertilize plants. The identity preservation submodule will express Rosea1, a transcription factor that activates anthocyanin synthesis pathways, leading to their accumulation, so that plants containing the biosafety module will be easily differentiated from non-transgenic plants.

FINAL PLANT

Finally, our purpose is to find an ideal plant which gathers the special characteristics to lay out the implementation of the plant as a real solution of the problem. This ideal plant is still to be determined, and will probably vary depending on the geographical situation of the crop.

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